Mesenteric lymph diversion abrogates 5-lipoxygenase activation in the kidney following trauma and hemorrhagic shock

Abstract

Background: Early acute kidney injury (AKI) following trauma is associated with multiorgan failure and mortality. Leukotrienes have been implicated both in AKI and in acute lung injury. Activated 5-lipoxygenase (5-LO) colocalizes with 5-LO-activating protein (FLAP) in the first step of leukotriene production following trauma and hemorrhagic shock (T/HS). Diversion of postshock mesenteric lymph, which is rich in the 5-LO substrate of arachidonate, attenuates lung injury and decreases 5-LO/FLAP associations in the lung after T/HS. We hypothesized that mesenteric lymph diversion (MLD) will also attenuate postshock 5-LO-mediated AKI.

Methods: Rats underwent T/HS (laparotomy, hemorrhagic shock to a mean arterial pressure of 30 mm Hg for 45 minutes, and resuscitation), and MLD was accomplished via cannulation of the mesenteric duct. Extent of kidney injury was determined via histology score and verified by urinary neutrophil gelatinase-associated lipocalin assay. Kidney sections were immunostained for 5-LO and FLAP, and colocalization was determined by fluorescence resonance energy transfer signal intensity. The end leukotriene products of 5-LO were determined in urine.

Results: AKI was evident in the T/HS group by derangement in kidney tubule architecture and confirmed by neutrophil gelatinase-associated lipocalin assay, whereas MLD during T/HS preserved renal tubule morphology at a sham level. MLD during T/HS decreased the associations between 5-LO and FLAP demonstrated by fluorescence resonance energy transfer microscopy and decreased leukotriene production in urine.

Conclusion: 5-LO and FLAP colocalize in the interstitium of the renal medulla following T/HS. MLD attenuates this phenomenon, which coincides with pathologic changes seen in tubules during kidney injury and biochemical evidence of AKI. These data suggest that gut-derived leukotriene substrate predisposes the kidney and the lung to subsequent injury.

Figure 1

Representative images of renal tubule histology in control, T/SS, T/HS, and MLD + T/HS rats. Normal renal tubule histology in control rat, with minimal loss of epithelial brush border, noncondensed nuclei, and minimal to no tubule collapse. T/SS kidney tubule histology demonstrating little brush border loss and some tubule collapse but with noncondensed nuclei. T/HS kidney shows severe tubular dilation (TD), tubular degradation, loss of all epithelial brush borders, nuclear collapse and nuclear loss, luminal congestion (LC), and tubular collapse (TC). MLD + T/HS kidney histology shows reversion to T/SS morphology, with none of the markers of tissue derangement seen in T/HS alone.

Figure 2

Histology score in control, T/SS, T/HS, and MLD +T/HS. Renal tubules (n = 100) from random HE kidney sections of the four groups were scored for tubule morphology on a scale from 0 to 3, with a maximum score generated for each kidney of 300; see Materials and Methods section). T/HS demonstrated global tubular damage, while MLD was protective. *p < 0.001.

Figure 3

NGAL measured in urine of control, T/SS, T/HS and MLD + T/HS rats. T/HS induces a significant increase in the AKI marker NGAL within 3 hours of end shock. MLD before hemorrhagic shock will reduce urinary NGAL levels; however, this did not reach statistical significance. *p < 0.05 compared with T/HS.

Figure 4

5-LO, FLAP and 5-LO/FLAP colocalization (FRET) in kidney. In all images, glycoprotein stain is gray. These images demonstrate FRET signal in a pseudocolor intensity scale. Increasing intensity of FRET signal results in a color spectrum change, with the most intense FRET signal displayed in red. This demonstrates the close associations between 5-LO and its cofactor FLAP in the T/HS group, which is not seen in any other cohort. The images also display total fluorescent intensities.

Figure 5

Total 5-LO/FLAP colocalization (FRET) intensity in control, T/SS, T/HS and MLD + T/HS, kidney images. FRET microscopy displayed in Figure 3. Total FRET signal (mean signal intensity × area) displayed in this figure. T/HS causes a significant increase in FRET signal versus control or T/SS, showing that FLAP and 5-LO are associated following T/HS. This signal is abrogated when mesenteric lymph is diverted before T/HS. ALUFI, arbitrary linear units of fluorescence intensity. *p < 0.001 versus all other groups.

Figure 6

Urinary cysteinyl leukotrienes in control, T/SS, T/HS, and MLD + T/HS. T/HS induces the increased excretion of 5-LO products in the urine. MLD will prevent this phenomenon from occurring. *p < 0.05 versus all other groups.